Paramyxoviruses are responsible for significant morbidity and mortality worldwide. Measles virus (MV), a member of the paramyxovirus family, accounts for approximately 600,000 deaths annually and is among the ten most lethal human pathogens. The virus is endemic in Africa, Asia and parts of Europe, and subject to frequent importation to the US. A herd immunity of approximately 95% is required to suppress periodic outbreaks. Despite high vaccination coverage in the US, cases are reported annually. No therapy is available for management of severe cases or rapid control of local outbreaks. It is therefore the long-term objective of this project to develop applicable antivirals that inhibit MV. Towards this goal a new class of small molecule MV fusion inhibitors has been identified that show strong promise as drug candidates. We propose structure-based design, pharmacophore extraction, quantitative structure-activity relationship (QSAR)-directed optimization and efficacy testing to develop this compound class to a therapeutic lead. To maximize the prospect of success and counteract viral resistance that may emerge in the field, additional, structurally unrelated drug candidates will be identified in parallel. The first specific aim is therefore to biochemically assess the target site of the fusion inhibitors and develop a three dimensional structure-based pharmacophore. Covalent photoaffinity labeling of the target protein by radiolabeled inhibitor analogs will be combined with protein microsequencing to define the physical binding site. The second specific aim identifies new hits through two complementary approaches: structure-guided database mining of available chemicals and high throughput screening using fluorescent MV as reporter. The third specific aim develops the fusion inhibitors and selected new hits to therapeutic leads through iterative rounds of QSAR-guided chemical modification and biotesting. Prior to lead development, new hits will be mechanistically characterized. The fourth specific aim is to assess the activity of selected leads against a representative panel of primary MV isolates that are frequently imported into the US and evaluate the efficacy of identified drug candidates in vivo using the cotton rat small animal model. Characteristic viral escape patterns will be identified for efficacious compounds that warrant future clinical development, and cross-resistance between structurally distinct therapeutic leads will be assessed.